Cocaine abuse is a major public health problem that directly or indirectly affects most communities and families. There is still no FDA-approved medication specific for treatment of cocaine dependence or overdose. Disastrous medical and social consequences of cocaine abuse have made the development of an anti-cocaine medication a high priority. Accelerating cocaine metabolism that produces biologically inactive metabolites via the most favorable cocaine-metabolizing pathway?cocaine hydrolysis catalyzed by human butyrylcholinesterase (BChE) in plasma?is recognized as the most efficient treatment strategy for cocaine overdose and dependence. Since the catalytic efficiency of wild-type BChE against the naturally occurring (-)- cocaine is low, we have designed and discovered a set of BChE mutants, known as cocaine hydrolases (CocHs), with at least 1,000-fold improved catalytic efficiency against (-)-cocaine compared to wild-type BChE. Preclinical and clinical data for the first one of our previously discovered CocHs has demonstrated the promise of enzyme therapy approach to the treatment of cocaine dependence. Our more recently designed and discovered novel CocH entity, denoted as CocH5-Fc(M6), which has not only further improved catalytic efficiency against cocaine, but also a considerably prolonged biological half-life. It has been demonstrated that a single dose of CocH5-Fc(M6) can be used to completely block cocaine-induced physiological, behavioral, and reinforcing effects for a long period of time in animal models. In addition, a stable CHO cell line capable of efficiently expressing CocH5-Fc(M6) and the corresponding master cell bank (MCB) have been developed along with establishment of the robust upstream and downstream protein production processes, ready for large-scale CocH5-Fc(M6) protein production. Built on the encouraging progress of our rational design, discovery, and development of the highly efficient, long-acting CocH entity CocH5-Fc(M6), the proposed new project is focused on further development of CocH5-Fc(M6) as a novel therapeutic candidate for cocaine dependence treatment, including large-scale production of the CocH5-Fc(M6) protein material using the developed MCB and established robust upstream and downstream protein production processes, investigational new drug (IND)-enabling studies, and first-in-human (FIH) clinical trials. The obtained clinical data about the safety, pharmacokinetics, and ex vivo pharmacodynamics of CocH5-Fc(M6) in humans will enable us to rationally design the most appropriate dosage regimen for future further clinical trials to determine the clinical efficacy of CocH5-Fc(M6) in cocaine-dependent patients. Thus, this investigation will move a promising candidate of the highly desired enzyme therapy closer toward FDA approval for cocaine dependence treatment.